Age related variations of some polymorphonuclear leukocyte functions

The age-related alterations of some biochemical processes in polymorphonuclear leukocytes (PMNL) obtained from 20 healthy aged male and 20 healthy aged female (age: 60-94 years) subjects were investigated. The basic level of luminol dependent chemiluminescence (LDCL) was increased, whereas the basal value of reduced/oxidized glutathione (GSH per GS-SG) was decreased in the aged groups. The enhancement of LDCL was more significant by PMNLs of aged males than of females. The change of both GSH and GS-SG levels during phagocytosis were diminished in the PMNLs of aged subjects. The L-alanine beta-naphthylamide specific elastase like protease (ELP) activity measured in living cell suspension was markedly increased with aging in male subjects. Therefore, it was concluded that the aging of PMNLs is partly a sex related process. The intensive release of both beta-glucuronidase (beta-G) and ELP by the PMNLs of aged subjects after in vitro treatment with calcium ionophore A23187, Cytochalasin B or low density lipoprotein (LDL) suggests that they could play an important role in some age-related disorders.     

Quantitation of polymorphonuclear leukocyte adherence to endothelial cells by electronic particle size discrimination

A method for quantitating the number of polymorphonuclear leukocytes (PMNs) adhering to endothelial cells in vitro is presented. Confluent human umbilical vein endothelial cells cultured in 24-well multiplates and treated with tumor necrosis factor (TNF ) were incubated subsequently wioth PMNs which adhere to the endothelial cell as a function of TNF concentration. Adherent PMNs and endothelial cells were proteolytically dissociated from the multiwell and, using an electronic particle counter, the number of endothelial cells and PMNs were determined simultaneously on the basis of size. The average number of PMNs adhering per endothelial cell was then calculated. The method is rapid and precise and offers an alternative to both tedious microscopic counting and the hazardous radiolabeling of PMNs.     

Modulation of human polymorphonuclear leukocyte function by the flavonoid silybin

The effect in vitro of the naturally occurring flavonoid silybin on human polymorphonuclear leukocyte (PMN) functions has been studied. Preincubation of PMNs for 10 min at 37 degrees C with silybin inhibited, in a dose-dependent way, the luminol-enhanced chemiluminescence (CL) generated by stimulated cells without affecting the non-enhanced CL or superoxide anion production evaluated by the cytochrome C reduction assay. No significant effect of silybin on PMN phagocytic or chemotactic activities were found. Silybin did not absorb light at the wavelength of luminol-enhanced CL and was not toxic to PMNs at the concentrations used. Catalase, a scavenger of H2O2, inhibited luminol-enhanced CL to a similar degree as silybin; moreover, when incubated together with PMNs, silybin and catalase did not produce an additive inhibition of CL. On the contrary, the simultaneous addition of silybin and sodium azide, an inhibitor of myeloperoxidase, further increased inhibition over that seen with azide alone. These results suggest that inhibition of H2O2 may be the mechanism by which silybin inhibits the luminol-enhanced CL generated by stimulated PMNs. Such results indicate a possible anti-inflammatory activity for silybin even if their clinical relevance remains to be elucidated.     

Enhancement of polymorphonuclear leukocyte (PMN) function by OK-432

The influence of OK-432, a streptococcal preparation, on human polymorphonuclear leukocytes (PMN) was examined. OK-432 increased O₂⁻ generation by PMN in oral cancer patients, and sustained the production of O₂⁻ in patients on chemoradiotherapy. Enhanced O₂⁻ generation was also observed when PMN were cultured with 10⁻² KE/ml OK-432 for 1 h and then stimulated with phorbol myristate acetate or formyl-metionyl-leucil-phenylalanine (FMLP). In addition, PMN O₂⁻ generation was promoted by culture supernatants of peripheral blood mononuclear cells (PBMC) incubated with 10⁻³ or 10⁻² KE/ml OK-432. Furthermore, OK-432 (10⁻³−10⁻² KE/ml) enhanced the chemiluminescence of FMLP- and PMA-stimulated PMN. However, nitroblue tetrazolium reduction and myeloperoxidase activity were only minimally enhanced. Not only the candidacidal activity of PMN but also antibody-dependent cell-mediated cytotoxicity against Candida and Raji cells were enhanced in correspondence with the increased generation of reactive oxygen species.Culture of PMN or PBMC for 24 h with OK-432 resulted in a concentration-dependent increase in the substantial production of interleukin-1β, interleukin-6 and tumor necrosis factor-α. OK-432 also enhanced granulocyte-macrophage colony stimulating factor and gamma-interferon generation by leukocytes in a dose-dependent manner. Our research indicates that OK-432 enhances PMN function directly as well as via the promotion of cytokine production, and suggests that these effects of OK-432 could be beneficial in immunosuppressed patients.     

Modulation of human polymorphonuclear leukocyte chemotaxis by leukocyte extracts,bacterial products,inflammatory exudates,and polyelectrolytes

Human polymorphonuclear leukocytes (PMN) chemotaxis was tested during exposure to leukocyte and platelet extracts, a variety of polyelectrolytes, inflammatory exudates, and bacterial products. The chemoattractants employed were either zymosan-activated serum or supernatant from autolyzedStaphylococcus aureus. Chemotaxis to both chemoattractants was markedly inhibited by leukocyte and platelet extracts; inflammatory exudates; anionic polyelectrolytes, DNA, hyaluronic acid, liquoid; and by cationic polyelectrolytes, histone, protamine base, protamine sulfate, and myeloperoxidase. Inhibition was also found with elastase, collagenase, pepstatin, and epsilon-aminocaproic acid. Bacterial products, such as lipoteichoic acid and lipopolysaccharides, and extracts of human dental plaque inhibited chemotaxis. No inhibition of chemotaxis was observed with heparin (<10 g/ml), chondroitin sulfate, phosphatidylethanolamine and phospatidylserine. Indeed, chondroitin sulfate markedly enhanced chemotaxis and antagonized the inhibitory effect of leukocyte or platelet extract. None of the agents employed was toxic to PMN as judged by trypan blue exclusion. These observations suggest that cationic polyelectrolytes and inflammatory exudates influence PMN surfaces, modifying interaction with chemoattractants. Assessment of the role of PMN chemotaxis inThis investigation was supported by research grants obtained through the courtesy of Dr. Samuel Robin of Cleveland, Ohio, the Alpha Omega Chapter, Friends of the Hebrew University in Manchester, England, and grant AI 08821 obtained from the National Institutes of Health for Dr. Paul G. Quie. Part of this investigation was performed at the Department of Pathology, School of Dental Medicine, University of Pennsylvania, Philadelphia supported by a research grants DE-02523, and DE-03995 from N.I.D.R.Dr. Isaac Ginsburg was the Lasby Visiting Professor at the University of Minnesota June–September, 1979, and a Visiting Professor at the University of Pennsylvania, October–December, 1979.Dr. Paul G. Quie is the American Legion Heart Research Professor.     

Clofazimine-mediated regulation of human polymorphonuclear leukocyte migration by pro-oxidative inactivation of both leukoattractants and cellular migratory responsiveness

The effects of clofazimine (0.15-20 micrograms/ml) on the spontaneous and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-stimulated migration, membrane-associated oxidative metabolism, degranulation and production of prostaglandin (PG) E2 by human polymorphonuclear in vitro have been investigated. Clofazimine at concentrations of 0.3 microgram/ml and greater significantly increased both the spontaneous and FMLP-stimulated chemiluminescence (CL), hexose monophosphate shunt (HMS) activity, myeloperoxidase-mediated protein iodination, auto-iodination, degranulation and PGE2 production by PMNL. At the same concentrations clofazimine inhibited both random and leukoattractant-induced migration of PMNL. Inhibition of PMNL migration by clofazimine was due to both a cell-directed auto-oxidative mechanism and by functional inactivation of FMLP. Clofazimine mediated inhibition of PMNL migration was prevented by the anti-oxidants cysteine and dapsone but not by the potent inhibitors of PG synthetase indomethacin and piroxicam. Anti-oxidants also protected FMLP from functional inactivation by clofazimine-exposed PMNL. Clofazimine increased both the spontaneous and FMLP-stimulated production of PGE2 by PMNL from four children with chronic granulomatous disease (CGD). Clofazimine is not an oxidising agent nor did it stimulate membrane-associated oxidative metabolism in CGD or NaF-pulsed normal PMNL. These data show that clofazimine-mediated inhibition of PMNL migration is dependent on intact cellular membrane-associated oxidative metabolism. Clofazimine is therefore a pro-oxidative anti-inflammatory agent.     

Influenza A virus enhances the human polymorphonuclear leukocyte chemoluminescence response without effecting inhibition by trifluoperazine

Influenza A virus has been demonstrated to enhance superoxide generation and chemoluminescence (CL) in human polymorphonuclear leucoytes (PMNs) under in vitro conditions. Although the mechanisms of virus-enhanced neutrophil activity is not established, calmodulin concentrations are known to increase in some virus-transformed cells. In the following experiments, we evaluated the PMN response to the calmodulin-inhibitor trifluoperazine (TFP) after an incubation with influenza A virus. Human PMNs were isolated from whole blood and were incubated with either influenza A virus (at 50% egg-infective dose per 1 leukocyte) or noninfected allantoic fluid. After incubation with influenza virus, the CL response of isolated PMNs to opsonized zymosan particles was measured. The influenza virus-treated PMNs had a mean (+/- SEM, n = 16) increase in light emission of 59.5 +/- 7.7%. TFP, in concentrations of 6 micron, 8 micron, and 10 microM, inhibition of CL was similar in influenza virus and allantoic fluid-treated neutrophils. These data suggest that, although the influenza A virus enhanced the PMN "respiratory burst" to opsonized zymosan particles, it did not alter the cell response to one calmodulin inhibitor, TFP.     

Modulation of human monocyte leukotactic responsiveness by thromboxane A2 and 12-hydroxyheptadecatrienoic acid (12-HHT)

The leukotactic responsiveness of human peripheral blood monocytes is regulated by the cell-directed inhibitor of monocyte leukotaxis, CDI-MLx. The actions of CDI-MLx on normal monocytes in vitro were abrogated by co-incubation with inhibitors of cyclooxygenase and thromboxane synthetase with indomethacin and dazmegrel (UK-38,485) being most active. The actions of CDI-MLx were mimicked by the thromboxane A2 analogue, U-46619, and by 12-HHT with half-maximal inhibition observed at 10(-10) M; PGE2 was 1000-fold less active. SQ 29,548, a thromboxane A2 receptor antagonist, blocked the effects of CDI-MLx, U-46619, and 12-HHT. Production of PGE2 and thromboxane B2 by purified monocytes was stimulated by CDI-MLx and this effect was also blocked by indomethacin, dazmegrel, and dazoxiben. These data suggest a major regulatory role for thromboxane synthetase products in human monocyte leukotaxis.     

Enhancement of human polymorphonuclear leukocyte adherence by the phospholipid mediator 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphorylcholine (AGEPC).

The phospholipid mediator 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphorylcholine (AGEPC) increases human polymorphonuclear leukocyte (PMN) adherence to Sephadex G-25 in manner directly proportional to the concentration of AGEPC from 2 to 650 nM. The maximal enhancement of PMN adherence of 85% +/- 23% (mean +/- SD) evoked by 650 nM AGEPC was indistinguishable in magnitude from that elicited by an optimal concentration of the chemotactic peptide N-f-Met-Leu-Phe. The results of analyses of the relative PMN adherence-enhancing activities of analogs of AGEPC suggest distinct molecular requirements for the enhancement of adherence. PMN adherence increases within 1 minute after exposure of the leukocytes to AGEPC, and the augmented adherence persists for over 30 minutes in spite of extensive washing of the leukocytes. Indomethacin partially blocked enhancement of adherence by AGEPC, suggesting that thromboxane A2 mediates in part the enhancement of adherence by AGEPC.     

Allergen-specific induction of interleukin-2 (IL-2) responsiveness in lymphocytes from children with asthma. II. Regulation of IL-2 responsiveness by supernatants of normal lymphocytes

Induction of interleukin-2 (IL-2) responsiveness by Dermatophagoides farinae (Df)-stimulated lymphocytes from children with asthma was markedly suppressed after the addition of culture supernatants from Df-stimulated normal T cells. Ovalbumin (OVA)-induced IL-2 responsiveness of lymphocytes from patients with hen-egg allergy was not suppressed by the supernatant from Df-stimulated T cells, indicating the antigenic specificity of the effect. Patients' lymphocytes whose IL-2 responsiveness was decreased still expressed Tac antigen (low-affinity IL-2 receptors) but, in contrast to the patients' original lymphocytes, did not absorb or respond to IL-2, suggesting the loss of high-affinity IL-2 receptors (p55/p75) from these cells. The supernatant from Df-stimulated normal T cells from one individual did not necessarily suppress the response of all patients tested, indicating the existence of some allogeneic barrier between the factor(s) and patients' lymphocytes.     

Interaction of inflammatory cells and oral microorganisms. II. Modulation of rabbit polymorphonuclear leukocyte hydrolase release by polysaccharides in response to Streptococcus mutans and Streptococcus sanguis.

The release of lysosomal hydrolases from polymorphonuclear leukocytes (PMNs) has been postulated in the pathogenesis of tissue injury in periodontal disease. In the present study, lysosomal enzyme release was monitored from rabbit peritoneal exudate PMNs exposed to Streptocccus mutans or Streptococcus sanguis. S. mutans grown in brain heart infusion (BHI) broth failed to promote significant PMN enzyme release. S. sanguis grown in BHI broth, although more effective than S. mutants, was a weak stimulus for promotion of PMN hydrolase release. Preincubation of washed, viable S. mutans in sucrose or in different-molecular-weight dextrans resulted in the ability of the organisms to provoke PMN release reactions. This effect could bot be demonstrated with boiled or trypsinized S. mutans or with viable S. sanguis. However, when grown in BHI broth supplemented with sucrose, but not with glucose, both S. mutans and S. sanguis triggered discharge of PMN enzymes. The mechanism(s) whereby dextran or sucrose modulates PMN-bacterial interaction may in some manner be related to promotion of microbial adhesiveness or aggregation by dextran and by bacterial synthesis of glucans from sucrose.     

Conversion of endogenous arachidonic acid to 5,15-diHETE and lipoxins by polymorphonuclear cells from patients with rheumatoid arthritis

The conversion of endogenous arachidonic acid (AA) by polymorphonuclear cells (PMN) from patients with rheumatoid arthritis (RA) was studied before (D0) and one day (D1) after antiinflammatory drug therapy. The biosynthesis of 5,15-diHETE and lipoxins (LXS), were investigatedex vivo, after PMN stimulation by ionophore A23187 without exogenous addition of 15-HETE. The eicosanoids were resolved by RP-HPLC and simultaneously detected at 246 and 302 nm respectively. Large amounts of 5,15-diHETE (50 to 400 ng/10⁷ PMN) and significant levels of LXS (from 2 to 20 ng/10⁷ PMN) were produced with individual differences between donors. Metabolite levels varied between patients but this work showed for the first time a linear relationship between the amounts of 5,15-diHETE and LXS. Moreover LXS production after treatment may be related to long-term clinical improvement of patients.     

Independent regulation of leukotriene B4-elicited polymorphonuclear leukocyte exocytosis and superoxide generation by a serum factor

Serum from a patient with inactive systemic lupus erythematosus (SLE) and ibuprofen-induced transient neutropenia was used as a probe to define further the control of human polymorphonuclear leukocyte (PMN) exocytosis and superoxide (O2-) generation. Thirty-minute preincubation of normal PMNs with 10-50% v/v of this serum, followed by washing, produced a specific dose-related suppression of leukotriene B4 (LTB4)-elicited beta-glucuronidase and lysozyme release of up to 45% and 30% respectively. If cells were not washed, the inhibition increased to 60% and 40%. Superoxide production stimulated by LTB4 was unaffected. The serum had no effect on formyl-met-leu-phe (FMLP) or phorbol myristate acetate-stimulated O2- or exocytosis. O2- and beta-glucuronidase release elicited by zymosan-treated serum (ZTS) were both decreased by 15%, but there was no increased inhibition seen if cells were not washed, or if the time of preincubation was increased from 7 to 30 min. In contradistinction, the serum inhibition of LTB4 exocytosis did show time dependence. Serum obtained when the patient was not leukopenic and sera from 6 normal controls, 2 patients with inactive SLE, 1 patient with SLE and chronic leukopenia, and 2 controls taking ibuprofen did not influence any PMN function. The serum inhibition of ZTS-induced functions was qualitatively similar to that observed when PMNs were preincubated and desensitized with ZTS in vitro. Selective inhibition of LTB4 exocytosis was not seen when PMNs were desensitized with LTB4 in vitro. These observations indicate that LTB4-elicited O2- and exocytosis can be independently and specifically regulated. The cellular site at which this serum factor acts is not clear, but the current studies strongly suggest that this inhibition is not due to in vitro deactivation by LTB4 activity.     

Modulation of leukotriene generation from human polymorphonuclear granulocytes by polychlorinated biphenyls (PCB).

Various isomers of polychlorinated biphenyls (PCB) were studied to induce and modulate the generation of lipo-oxygenase products (leukotrienes) from human polymorphonuclear granulocytes (PMN) under non-cytotoxic conditions. Stimulation of human PMN with PCB did not induce leukotriene generation by themselves, but modulated the subsequent leukotriene formation when opsonized zymosan or sodium fluoride (NaF) were applied as stimuli. The simultaneous addition of PMN with opsonized zymosan and PCB, especially 3,3',4,4'-TCB, led to an increased generation of leukotrienes compared to the control. 3,3',4,4'-TCB exceeded the activity of the remainder PCB in a dose-dependent manner and also induced a chemiluminescence response from PMN. The simultaneous incubation of PMN with NaF and 3,3'-DCB,3,3',4,4'-TCB and 2,2',3,3'-TCB showed an inhibitory effect on leukotriene generation unlike NaF stimulation in the absence of PCB. Incubation of PMN with PCB had no significant effect on leukotriene generation induced by the Ca-ionophore (5 microM). Our data show a direct relationship between the extent of PMN stimulation and the chloro-substitution pattern of the PCB in combination with the different stimuli.